The present invention relates to a process of producing a heterologous protein, especially transglutaminase, by secretory production. The heterologous proteins produced by the method include industrially useful enzymes, physiologically active proteins and others. Transglutaminase has been widely used for food processing, the manufacture of pharmaceuticals and the like.
A number of processes for the secretory production of heterologous proteins have been previously reported including those as described in the review on the secretory production of a heterologous protein by a bacterium belonging to the genus Bacillus [Microbial. Rev., 57, 109-137 (1993)], the review on the secretory production of a heterologous protein by methylotrophic yeast Pichia pastoris [Biotechnol., 11, 905-910 (1993)] and the report on the industrial production of heterologous proteins by the mould belonging to the genus Aspergillus [Biotechnol., 6, 1419-1422 (1988); Biotechnol., 9, 976-981 (1991)].
The transglutaminase produced by the secretory production according to one embodiment of the present invention is an enzyme which catalyzes acyltrasfer reaction of γ-carboxylamide groups in the peptide chain of the protein. When the enzyme is reacted with a protein, the formation of the cross-linkage ε-(γ-Glu)-Lys and the replacement of Gln with Glu by deamidation can be occurred. Transglutaminase has been used to manufacture gelled food products such as jelly, yogurt, cheese or gelled cosmetics and others, and to improve the quality of meat, etc (Japanese publication of examined application No. 1-50382). Moreover transglutaminase is an enzyme having industrially high usefulness in that it has been used to manufacture materials for thermostable microcapsules, carriers for immobilized enzymes etc.
Transglutaminases derived from animals and from microorganisms (microbial transglutaminase: referred to as ‘MTG’ hereinafter) have been previously known. The former is the calcium ion-dependent enzyme which is distributed in animal organs, skin, blood, etc. The examples include guinea pig hepatic transglutaminase (K. Ikura et al. Biochemistry 27, 2898 (1988)), human epidermal keratinocyte transglutaminase (M. A. Phillips et al. Proc. Natl. Acad. Sci. USA 87, 9333 (1990)), human blood coagulation factor XIII (A. Ichinose et al. Biochemistry 25, 6900 (1990)) and others.
For the latter, calcium-independent transglutaminases have been discovered from bacteria belonging to the Streptoverticillium genus, which include, for example, Streptoverticillium griseocarneum IFO 12776, Streptoverticillium cinnamoneum sub sp. cinnamoneum (hereinafter it can be abbreviated as S. cinnamoneum) IFO 12852, Streptoverticillium mobaraense (hereinafter it may abbreviated as S. mobaraense) IFO 13819 and others (Publication of unexamined Japanese patent application (JP-Kokai) No. 64-27471). The peptide mapping and the structural analysis of the genes revealed that the primary structure of the transglutaminase produced by these microorganisms shared no homology with transglutaminases from animals (European Patent application No. 0 481 504 A1).
Because microorganism-derived transglutaminase (MTG) is produced through the purification from the cultures of microorganisms such as described above, there have been problems in terms of the amount and the efficiency and the like. The production of transglutaminase using genetically engineered procedure has been also attempted. Transglutaminase proteins and the genes thereof have been reported in, for example, Biosci. Biotechnol. Biochem., 58, 82-87 (1994), Biosci. Biotechnol. Biochem., 58, 88-92 (1994), Biochimie, 80, 313-319 (1998)., Eur. J. Biochem., 257, 570-576 (1998), WO 96/06931, WO 96/22366, etc, which report the expression and production of transglutaminase in host-vector systems such as Streptomyces lividans, Aspergillus oryzae and Escherichia coli. In addition to these information, a process wherein transglutaminase is produced by secretory production in microorganisms such as E. coli and yeast (JP-Kokai No. 5-199883) and the method has been reported wherein MTG having activities is produced by expressing MTG as an inactive fused protein in an inclusion body within E. coli and subsequently solubilizing the inclusion body using protein-denaturing agents, and then, reconstituting it through the removal of the denaturing agents (JP-Kokai No. 6-30771). However, the problem has been noted that the expression level is significantly low in the secretory production by microorganisms such as E. coli or yeast.
On the other hand, there are examples of previous studies for the efficient secretory production of heterologous proteins using a coryneform bacterium include the secretion of nucleases and lipases [U.S. Pat. No. 4,965,197, J. Bacteriol., 174, 1854-1861 (1992)] and the secretion of proteases such as subtilisin [Appl. Environ. Microbiol., 61, 1610-1613 (1995)] by Corynebacterium glutamicum (hereinafter it may be abbreviated as C. glutamicum), a study on the secretion of cell surface proteins of a coryneform bacterium [International patent application published in Japan No. Hei 6-502548], the secretion of fibronectin-binding protein using this study [Appl. Environ. Microbial., 63, 4392-4400 (1997)], a report wherein the secretion of proteins was enhanced using a mutated secretory machinery [JP-Kokai No. 11-169182], etc., but there has been a limited number of reports on limited proteins. In light of the accumulated amount of proteins, Appl. Environ. Microbial., 61, 1610-1613 (1995) describes that about 2.5 mg/ml of protein was accumulated by expressing the alkaline protease gene from Dichelobacter nodosus within C. glutamicum using a promoter of subtilisin gene (aprE) from Bacillus subtilis, ribosome binding site and the sequence of a signal peptide, but U.S. Pat. No. 4,965,197, JP-Kokai No. 6-502548 or JP-Kokai No. 11-169182 do not specifically describe the values of the amount of the proteins secreted and accumulated. Furthermore, in the case of the fibronectin-binding protein [Appl. Environ. Microbiol., 63, 4392-4400 (1997)], only the secretory accumulation of the protein of about 2.5 μg/L is confirmed. Thus, there has been no reports that heterologous proteins could be efficiently accumulated in the medium at a practical level.
Additionally a genetic engineering technology for a coryneform bacterium has been developed in the system using plasmid and phage, such as the establishment of the transformation by protoplast [J. Bacteriol., 159, 306-311 (1984); J. Bacteriol., 161, 463-467 (1985)], the development of a various type of vectors [Agric. Biol. Chem., 48, 2901-2903 (1984); J. Bacteriol., 159, 306-311 (1984); J. Gen. Microbiol., 130, 2237-2246 (1984); Gene, 47, 301-306 (1986); Appl. Microbiol. Biotechnol., 31, 65-69 (1989)], the development of the regulation method of gene expression [Bio/Technology, 6, 428-430 (1988)] and the development of cosmid [Gene, 39, 281-286 (1985)]. Moreover there are reports on the cloning of genes from a coryneform bacterium [Nucleic Acids Res., 14, 10113-1011 (1986); J. Bacteriol., 167, 695-702 (1986); Nucleic Acids Res., 15, 10598 (1987); Nucleic Acids Res., 15, 3922 (1987); Nucleic Acids Res., 16, 9859 (1988); Agric. Biol. Chem., 52, 525-531 (1988); Mol. Microbiol., 2, 63-72 (1988); Mol. Gen. Genet., 218, 330-339 (1989); Gene, 77, 237-251 (1989)].
Further a transposable element derived from a coryneform bacterium has also been reported [WO93/18151; EPO445385; JP-Kokai No. 6-46867; Mol. Microbiol., 11, 739-746 (1994); Mol. Microbiol., 14, 571-581 (1994); Mol. Gen. Genet., 245, 397-405 (1994); FEMS Microbiol. Lett., 126, 1-6 (1995); JP-Kokai No. 7-107976].
The transposable element means a DNA fragment which can be transposed on the chromosome and is known to be present in a wide range of organisms ranging from prokaryotes to eukaryotes. Transposons using transposable elements have been developed [WO93/18151; JP-Kokai No. 7-107976; Mol. Gen. Genet., 245, 397-405 (1994); JP-Kokai No. 9-70291] and a heterologous gene has become to be able to be expressed using a transposon.